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Host cell protein networks as a novel co-elution mechanism during protein A chromatography.
No full textHost cell proteins (HCPs) are process-related impurities of therapeutic proteins produced in for example, Chinese hamster ovary (CHO) cells. Protein A affinity chromatography is the initial capture step to purify monoclonal antibodies or Fc-based proteins and is most effective for HCP removal. Previously proposed mechanisms that contribute to co-purification of HCPs with the therapeutic protein are either HCP-drug association or leaching from chromatin heteroaggregates. In this study, we analyzed protein A eluates of 23 Fc-based proteins by LC-MS/MS to determine their HCP content. The analysis revealed a high degree of heterogeneity in the number of HCPs identified in the different protein A eluates. Among all identified HCPs, the majority co-eluted with less than three Fc-based proteins indicating a drug-specific co-purification for most HCPs. Only ten HCPs co-purified with over 50% of the 23 Fc-based proteins. A correlation analysis of HCPs identified across multiple protein A eluates revealed their co-elution as HCP groups. Functional annotation and protein interaction analysis confirmed that some HCP groups are associated with protein-protein interaction networks. Here, we propose an additional mechanism for HCP co-elution involving protein-protein interactions within functional networks. Our findings may help to guide cell line development and to refine downstream purification strategies
People of TM: Videos of Eshita Khera
No full textThese videos will be used for external social media engagement campaign on platforms like LinkedIn and YouTube etc.featuring stories of people in TM. No IP related content
Viral Kinetics Model of SARS-CoV-2 Infection Informs Drug Discovery, Clinical Dose, and Regimen Selection.
No full textQuantitative systems pharmacology (QSP) has been an important tool to project safety and efficacy of novel or repurposed therapies for the SARS-CoV-2 virus. Here, we present a QSP modeling framework to predict response to antiviral therapeutics with three mechanisms of action (MoA): cell entry inhibitors, anti-replicatives, and neutralizing biologics. We parameterized three distinct model structures describing virus-host interaction by fitting to published viral kinetics data of untreated COVID-19 patients. The models were used to test theoretical behaviors and map therapeutic design criteria of the different MoAs, identifying the most rapid and robust antiviral activity from neutralizing biologic and anti-replicative MoAs. We found good agreement between model predictions and clinical viral load reduction observed with anti-replicative nirmatrelvir/ritonavir (Paxlovid®) and neutralizing biologics bamlanivimab and casirivimab/imdevimab (REGEN-COV®), building confidence in the modeling framework to inform a dose selection. Finally, the model was applied to predict antiviral response with ensovibep, a novel DARPin therapeutic designed as a neutralizing biologic. We developed a new in silico measure of antiviral activity, area under the curve (AUC) of free spike protein concentration, as a metric with larger dynamic range than viral load reduction. By benchmarking to bamlanivimab predictions, we justified dose levels of 75, 225, and 600 mg ensovibep to be administered intravenously in a Phase 2 clinical investigation. Upon trial completion, we found model predictions to be in good agreement with the observed patient data. These results demonstrate the utility of this modeling framework to guide the development of novel antiviral therapeutics
Multiple Genes Core to ERAD, Macroautophagy and Lysosomal Degradation Pathways Participate in the Proteostasis Response in α1-Antitrypsin Deficiency.
No full textIn the classic form of α1-antitrypsin deficiency (ATD), the misfolded α1-antitrypsin Z (ATZ) variant accumulates in the endoplasmic reticulum (ER) of liver cells. A gain-of-function proteotoxic mechanism is responsible for chronic liver disease in a subgroup of homozygotes. Proteostatic response pathways, including conventional endoplasmic reticulum-associated degradation and autophagy, have been proposed as the mechanisms that allow cellular adaptation and presumably protection from the liver disease phenotype. Recent studies have concluded that a distinct lysosomal pathway called endoplasmic reticulum-to-lysosome completely supplants the role of the conventional macroautophagy pathway in degradation of ATZ. Here, we used several state-of-the-art approaches to characterize the proteostatic responses more fully in cellular systems that model ATD.We used clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genome editing coupled to a cell selection step by fluorescence-activated cell sorter to perform screening for proteostasis genes that regulate ATZ accumulation and combined that with selective genome editing in 2 other model systems.Endoplasmic reticulum-associated degradation genes are key early regulators and multiple autophagy genes, from classic as well as from ER-to-lysosome and other newly described ER-phagy pathways, participate in degradation of ATZ in a manner that is temporally regulated and evolves as ATZ accumulation persists. Time-dependent changes in gene expression are accompanied by specific ultrastructural changes including dilation of the ER, formation of globular inclusions, budding of autophagic vesicles, and alterations in the overall shape and component parts of mitochondria.Macroautophagy is a critical component of the proteostasis response to cellular ATZ accumulation and it becomes more important over time as ATZ synthesis continues unabated. Multiple subtypes of macroautophagy and nonautophagic lysosomal degradative pathways are needed to respond to the high concentrations of misfolded protein that characterizes ATD and these pathways are attractive candidates for genetic variants that predispose to the hepatic phenotype
Author Correction: The state of the art in secondary pharmacology and its impact on the safety of new medicines.
No full textsame as for 4937
Deep resolution of clinical, cellular, and transcriptomic inflammatory markers during 52 weeks of IL-17A inhibition by secukinumab
No full textBackground
Secukinumab, an anti-IL-17A monoclonal antibody, induces histological and molecular resolution of psoriatic plaques by 12 weeks. However, the long-term effects of secukinumab on molecular resolution of psoriatic inflammation remain unknown.
Objective
To investigate the molecular resolution of psoriasis following 52-weeks of secukinumab treatment.
Methods
NCT01537432 was a two-part Phase 2, randomised, double-blinded, placebo-controlled, 52-week study of patients with moderate to severe psoriasis receiving secukinumab 300 mg. Psoriatic lesional and non-lesional skin biopsies were obtained at baseline, Week 12, and Week 52, and the composition of the residual disease genomic profile (RDGP, i.e., “molecular scar”) of biopsies from secukinumab-responders was analysed.
Results
After 52 weeks of treatment, 14/24 enrolled patients were considered clinical responders (≥75% improvement in Psoriasis Area and Severity Index [PASI]; PASI75), 4/24 were considered non-responders (<PASI75), and 6/24 patients were lost to follow-up; both histological and transcriptomic profiles of PASI75 responders improved from Week 12–52. RDGP transcripts of histological responders only partially overlapped between Week 12 and 52, despite similar number of transcripts in each RDGP; specifically, four novel transcript subsets showed distinct expression dynamics between Week 12 and 52 (slow-resolving, recurring, persistent, and resolved), with anti-inflammatory and immunomodulatory genes (e.g., SOCS1, CD207, IL-37) notably restored at Week 52. Shorter disease duration prior to secukinumab treatment coincided with greater transcript improvements at Week 12 and Week 52.
Conclusions
Secukinumab improves the histological and molecular phenotype of psoriatic lesional skin up to 52 weeks of treatment; these results suggest possible mechanisms that drive long-term control of psoriasis
Chemoenzymatic Synthesis of 2-Aryl-thiazolines fr
No full textNitrogen heterocycles are commonly found in bioactive
natural products and drugs. However, the biocatalytic tools for
nitrogen heterocycle synthesis are very limited. Herein, we report our
discovery of vanillyl alcohol oxidases (VAOs) as an efficient
biocatalyst for the one-pot synthesis of 2-aryl-thiazolines from 4-
hydroxybenzaldehydes and aminothiols. The wild-type biocatalyst is
featured with broad scope of 4-hydroxybenzaldehydes. Though the
scope of aminothiols is limited, it can be overcome via semi-rational
protein engineering, generating a variant that can be used to produce
previously inaccessible cysteine-derived bioactive 2-aryl-thiazolines
using the wild-type VAO. Benefiting from the derivatizable functional
groups in the enzymatic products, these products can be chemically
modified to further expand the chemical space that can be covered by
VAO, offering a new chemoenzymatic strategy for green and
convenient synthesis of structurally diverse 2-aryl-thiazoline
derivatives in drug development
Double-stranded RNA induces retinal pigment epithelium cell degeneration and inflammation
No full textRIG-I signaling has been previously implicated as a driver of inflammation to the retinal pigment epithelium (RPE) during age-related macular degeneration (AMD). Double-stranded RNA (dsRNA) is known to initiate RIG-I signaling and lead to a type I interferon response. We show through shRNA knockdown that RIG-I is essential for initiating an interferon response in iPS-RPE in response to both synthetic dsRNA-mimetic 3p-hpRNA and the double-stranded retrotransposable element Alu. Analysis of human tissue from patients suffering from AMD show accumulation of dsRNA, peaking at the geographic atrophy (GA) stage. Using a new murine model of 3p-hpRNA subretinal challenge to RPE cells, we confirmed that accumulation of dsRNA initiates a type I interferon response, as well as RPE and photoreceptor degeneration. Although RPE response to synthetic dsRNA was acute, extensive leukocyte migration was observed. The results from this study verify the importance of RIG-I signaling in regulating inflammation in the subretinal space and implicates dsRNA accumulation as a driver of AMD pathogenesis
Tolerability of intravenous administration of porous silicon nanoparticles in mice
No full textPorous silicon nanoparticles (pSiNP), obtained from our external collaborator Nacamed, were administered intravenously to wild type mice to assess their tolerability before using this route of administration in the presence of immunomodulatory compounds All dosed groups tolerated the pSiNP and no clinical signs were observed at any time point